This invention relates to a novel strain of an alkalothermophilic Bacillus sp. More particularly the invention relates to a process for the preparation of a low molecular weight peptidic aspartic acid protease inhibitor using a novel strain of alkalothermophilic Bacillus (AT Bacillus sp.) deposited at American Type Culture Collection (ATCC) and bearing Accession Number PTA-972.
Proteases are responsible either directly or indirectly for all bodily functions including cell growth, nutrition, differentiation and apoptosis. They also play a significant role in intracellular and extracellular protein turn over (house keeping and repair), cell migration and invasion, fertilization and implantation (Protease inhibitors, novel therapeutic application and development, Tony E Hugli, TIBTECH, 14, 409-412, 1996). Since proteases are necessary for normal and abnormal body functions, their effective regulatory counterparts i.e., protease inhibitors, are tremendously essential for physiological regulations. Protease inhibitors have been the source of attention in many disciplines. Due to their presence in valuable plant feeds and involvement in nutritive properties they have evoked the interest of nutritionists. Inhibitor proteins have been studied for the elucidation of mechanism of inhibition of proteases, as well as for the studies on protein-protein interactions and associations. Due to their unique pharmacological properties, protease inhibitors are also used as valuable tools in medical research.
Protease inhibitors are classified into Synthetic and Naturally occurring inhibitors. They occur in numerous animal tissues and fluids, in many plant tissues (particularly in legume seeds and other storage organs) and in microorganisms (Protease inhibitors, Yehudith Birk, Hydrolytic enzymes, A Neuberger and K. Brocklehurst (Eds), Elsevier Science Publishers B. V. (Biomedical Division), 257, 1987). The most abundant source of the inhibitors in plants is the seeds, but their location is not necessarily restricted to this part of the plant. They are also found in leaves, tubers, etc. As for the intracellular localization of the inhibitors, they appear to be associated primarily with the cytosol, but in some instances they have been localized in protein bodies. The inhibitors of animal origin are found both in tissues and in secretions of organs. The pancreatic trypsin inhibitor has been found as an intracellular component in various bovine organs: in the pancreas, lung, liver, spleen, paratoid gland and also in pituitary gland. In addition to the thoroughly studied pancreatic trypsin inhibitors, a large number of protease inhibitors from different animal sources have been isolated. Many of them are secretory proteins, such as trypsin inhibitors of blood plasma, milk colustrum, seminal plasma and submandibular glands. The plasma protease inhibitors constitute a major group of the functional proteins of the blood plasma. Most of them inhibit serine proteases but their mechanism of interaction is still being actively pursued by many investigators.
The presence of protease inhibitors in microorganisms came into existence from the studies on antibiotics as they act as inhibitors of the enzymes which are involved in growth and multiplication. Proteolytic enzymes outside of microbial cells hydrolyze organic nitrogen compounds in the medium, so they are thought to be harmful to cells. The production of inhibitors of the proteolytic enzymes by microorganisms is probably a mechanism to provide cell protection. In contrast to the inhibitors of proteolytic enzymes obtained from animals and plants, the inhibitors from microorganisms are of smaller molecular nature. Specific inhibitors of microbial origin have been used as useful tools in biochemical analysis of biological functions and diseases. (Enzyme inhibitors of microbial origin, Hamao Umezawa, University Park Press).
A few of the inhibitors of microbial origin of therapeutic interest are given below:
Leupeptin-from Streptomyces, is the inhibitor of trypsin, plasmin, kalikrein and papain.
Chymostatin-from Streptomyces, is the inhibitor of chymotrypsin.
Dopastin-from Pseudomonas, Oosponol-from Oospora, Oudenone-from Oudemansialla radicata and Fusaric acid-from Fusarium are the inhibitors of dopamine hydroxylase.
Pepstatin Axe2x80x94from Streptomyces, is the inhibitor of pepsin, an aspartic acid protease. It inhibits the HIV-1 protease, which is also an aspartic protease and the key enzyme for the propagation of the HIV.
The expanding Acquired Immuno Deficiency Syndrome (AIDS) epidemic and the relentless nature of the disease have intensified the search for effective antiviral therapies, to control the replication of the HIV, the causative agent of AIDS. The HIV-1 protease is the key enzyme for the propagation of the virus. Thus specific inhibition of the HIV-1 protease by inhibitors is useful in preventing the infection HIV-1 protease is structurally and mechanistically related to mammalian and microbial aspartic proteases such as pepsin, cathepsin, renin, and endothiopepsin. The classification of HIV-1 protease in the aspartyl family was also predicted from its primary sequence analysis. A highly conserved sequence Asp-Thr-Gly (D-T-G) in retroviral proteases, is also conserved in the active site of the cellular and fungal proteases. Molecular modeling studies have also confirmed the functional and structural similarities of the retroviral proteases to other aspartyl proteases. Various synthetic peptide and non-peptide compounds have been shown to inhibit HIV-1 protease. Well documented examples of isolation of compounds by microbial screening represented by the discovery of potent compounds such as cyclosprin, movionolin and avermycin, etc. An antifungal antibiotic cerulenin from Cephalosporium and pepstatin A, a pepsin inhibitor from Streptomyces, have been well characterized as HIV-1 protease inhibitors (C.Debouck, AIDS Research and Human Retroviruses, 8, 153-164, 1992).
Extensive evidence suggests that, the degradation of hemoglobin is necessary for the growth of erythrocytic malarial parasite, apparently to provide free amino acids for parasitic protein synthesis. On the basis of the data available, the aspartic acid proteases are thought to be responsible for the initial cleavages of hemoglobin. Both aspartic acid and cystein proteases have synergistic effects in inhibiting the growth of the cultural malarial parasite and also these proteases act synergistically to degrade hemoglobin. Therefore, the combination of inhibitors of malarial cystein and aspartic acid proteases, may provide a most effective chemotherapeutic regimen and best limit the development of parasitic resistant to protease inhibitors. Pepstatin, the inhibitor of aspartic acid proteases, along with the cystein protease inhibitor E-64, blocks the Plasmodium falciparum development. (Proteases of Malarial Parasite: New Targets for Chemotherapy, Philip J. Rosenthal, Emerging Infectious Diseases, 4(1), 49-57, 1998). So far no report is available for the preparation of the protease inhibitor using alkalothermophilic Bacillus sp.
Based on the fact that the aspartic acid protease plays a significant role in the development of the malarial parasite, the applicants believe that the inhibitor produced in accordance with the practice of the invention using the novel strain of Bacillus sp deposited at ATCC having Accession No. PTA 972, could be a potent inhibitor for proteases, particularly aspartic acid protease, and more particularly for proteases of malarial parasites. The inhibitor described in the present invention inhibits pepsin, an aspartic protease. Pepsin present in the gastric secretion is responsible for the degradation (digestion) of proteinaceous food. Excess secretion of pepsin has harmful effects on the stomach as it damages the digestive tract and causes stomach ulcer or duodenal ulcer. Considering the fact that the inhibitor is an active inhibitor of pepsin, it has potential application as a therapeutic agent against stomach or duodenal ulcers. Pepstatin A, a pepsin inhibitor has been reported to inhibit HIV-1 protease which is also an aspartic protease. The applicants have observed that the inhibitor also inhibits other enzymes having aspartic acid in the active site, and felt that the microbial protease inhibitor could inhibit HIV-1 protease.
The main objective of the present invention is to provide a process for the preparation of low molecular weight peptidic aspartic acid protease inhibitor using a novel alkalothermophilic strain of Bacillus sp. deposited at American Type Culture Collection, and having Accession number PTA 972.
Another object of the invention is to develop an anti-fungal agent using the low molecular weight aspartic acid protease inhibitor developed in accordance with the process of the invention.
The invention provides a novel alkalothermophilic strain of Bacillus sp. deposited at ATCC and bearing Accession Number PTA 972. The invention also provides a process for the preparation of a low molecular weight aspartic acid protease inhibitor using the said novel strain and Bacillus sp.
Accordingly, the present invention provides a process for the preparation of low molecular weight peptide aspartic acid protease inhibitor using an alkalothermophilic Bacillus sp. deposited at ATCC and having Accession Number PTA 972, said method comprising the steps of:
(i) subculturing the alkalothermophilic Bacillus sp bearing Accession Number PTA 972 in a fermentation medium at a temperature in the range of 37xc2x0 C. to 50xc2x0 C. under aseptic conditions for a period ranging between 24 to 48 hrs.,
(ii) harvesting the culture broth,
(iii) separating the solids by conventional methods to obtain cell free liquid containing the protease inhibitor,
(iv) treating the cell free liquid with a decolorising agent for a period ranging between 2 to 24 hrs to obtain a colorless liquid,
(v) separating the decolorising agent and recovering the protease inhibitor by conventional methods.
The strain of alkalothermophilic Bacillus sp. employed in the present invention was isolated from the hot spring from Vajreshwari, District Thane, State of Maharashtra, India and has been deposited at the National Collection for Industrial Micro-organisms, NCL, Pune 411 008, India at Accession No. NCIM 59. The said strain of Bacillus sp. is also deposited at American Type Culture Collection and bears accession number PTA-972. The range of pH for the growth of the isolate was alkaline from 8-10 with an optimum growth at pH-10. No growth occurred below pH-7.0. The temperature range for the growth was 37-50xc2x0 C. The strain was characterized as an aerobic, gram-positive, motile, spore-forming bacterium. On alkaline nutrient agar medium at 50xc2x0 C., the colonies were butyrous, glistening and pale cream colored. The strain showed negative reaction towards production of indole, hydrogen sulfide, ammonia and urease, but positive reactions for the hydrolysis of starch, for the Voges-Proskuaer test, for the production of the catalase, for the hydrolysis of casein and for the reduction of nitrate. The organism produced oxidase, and ammonia was utilised as a nitrogen source. Acid but no gas was produced aerobically or anaerobically from D-glucose, D-mannose, D-fructose, and sucrose.
In an embodiment, the fermentation medium comprises assimilable carbon and nitrogen sources, malt extract, yeast extract, peptone, and micro-ingredients. The medium further comprises:
In still another embodiment, assimilable carbon sources in the fermentation medium are selected from the group consisting of lactose, sorbitol, xylan, fructose, maltose and sucrose and the nitrogen sources are selected from the group consisting of soyameal, casein, casamino acids, urea, tryptone, beef extract, skimmed milk and yeast extract.
In a further embodiment, the protease inhibitor is recovered by treating the cell free supernatant with decolorizing agent, separating the low molecular weight compound from the supernatant by conventional methods selected from ultrafiltration, concentrating the filtrate by lyophilization, and purifying the inhibitor by reverse phase high performance liquid chromatography.
In an embodiment, the decolorizing agent, is selected from activated charcoal or cellulose.
In another embodiment, the production of the inhibitor and its activity is also dependent upon the nature and concentration of the media ingredients, inoculum development and parameters such as aeration, agitation, etc. In shake flasks maximum production was obtained after 24-48 hours, when a 5-20% v/v vegetative inoculum in the actively growing phase is transferred to the production media. Post fermentation processing of the broth for the isolation of the inhibitor includes, centrifugation or filtration of the broth. Cell free supernatant is treated with activated charcoal to remove the color material. The charcoal is removed by centrifugation or filtration. The resulting supernatant is subjected to ultrafiltration using filtration membranes.
In a feature of the invention, the pepsin inhibitor was selectively purified by removing the high molecular weight compounds from the culture broth by treating it with activated charcoal. The charcoal treated broth was filtered through ultrafiltration membrane and concentrated by lyophilization. The concentrated inhibitor sample was loaded onto a reverse phase-high performance liquid chromatography column using a linear gradient of acetonitrile and triflouroacetate, and the peaks were checked for the inhibitor activity. Among the two compounds detected in the HPLC analysis, the compound having less retention time showed the inhibition against pepsin. This compound absorbs strongly at 210 nm. The compound was collected and checked as a single purified peptide after loading onto the HPLC column. One unit of inhibitor is defined as the amount of inhibitor which inhibits the protease activity expressed in terms of decrease in optical density of 0.001 per minute.
Without being bound by any theory, the Applicants hereby state that the alkaline protease inhibitor developed in accordance with the process of the invention exhibits anti-fungal properties against a wide spectrum of fungi such as Aspergillus sp, Altemaria sp, Fusarium sp and Trichoderma sp., and is therefore useful as an anti-fungal agent.